The experiments described in this proposal are designed to examine the requirements for recognition of determinants encoded (or qualitatively regulated) by the H-2 unlinked Mls locus. A strong primary in vitro response (T cell proliferation and lymphokine production) to Mls incompatible stimulator cells is observed. This response is reflected in a high frequency of specifically reactive precursor cells (approximately 10-fold higher than for allo-H-2). Recent findings of ours, in a tolerance system, strongly suggest that Mls determinants are recognized as true allo-antigens. However, the immunological role of these antigens remains a matter of speculation due, to a large degree, to the lack of conclusively specific serological reagents. It is hoped that the experiments described in the last section of the grant application will also characterize the biochemical nature of these Mls determinants and lead to the generation of serological reagents specific for them. (1) Experiments are planned to map the H-2 linked genes determining our previously demonstrated capacity to stimulate an anti-Mls response and to define the role of Ia antigens in the response to Mls antigens. Using the Southern blot technology, we will examine Mls specific T-T hybrids for potential M1s specific T cell receptor gene rearrangements. (2) Protocols aimed at analysing Mls specific tolerance in terms of the Mls antigen presentation requirements for its induction and the specificity between the various Mls haplotypes of the tolerant state are described. (3) Parallel approaches, using an artificial membrane methodology and xenogeneic monoclonal antibody production, will be pursued in an attempt to characterize an Mls antigen. It is hoped that the experiments outlined in this proposed 3-year study will provide information and serological reagents that will facilitate a subsequent molecular biological analysis of the Mls locus and an investigation of the functional role of its products.s